Anti-La antibodies and their use for immunotargeting

ABSTRACT

The invention relates to antibodies against the human La protein and to their use in immunotargeting, in particular the immunotargeting of tumor cells. The object of the invention is to provide improved antibodies which bind universal target structures on the surface of tumor cells, and to provide novel anti-La antibodies, in particular with a high affinity for La, a universal target structure on tumor cells, which make it possible to use the antibodies as recombinant fragments for immunotargeting. The invention comprises recombinant antibodies comprising: (i) a binding unit of an antibody which specifically binds to an epitope of a human nuclear antigen, preferably human La protein, and (ii) a binding unit of an antibody which specifically binds to an effector cell or of a ligand which specifically binds to an effector cell. The invention furthermore comprises novel antibodies which specifically bind the human La protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/814,510, filed May 3, 2013, which is a U.S. National Phase filingunder 35 U.S.C. § 371 of International Application No.PCT/EP2011/063539, filed Aug. 5, 2011, which claims benefit of priorityto German Patent Application No. 10 2010 039 018.6, filed Aug. 6, 2010,the entire disclosures of which are hereby incorporated by referenceherein.

BACKGROUND OF THE INVENTION

The invention concerns antibodies against the human La protein and theiruse for immunotargeting, in particular of tumor cells. The antibodiesaccording to the invention are suited for use in the field of medicine,pharmacy and in biomedical research.

The human La protein (hLa) was originally disclosed as an auto-antigenin patients with systemic lupus erythematosus (SLE, Mattioli, M.,Reichlin, M., 1974. Arthritis & Rheumatism; 17 (4): 421-29) and Sjögrensyndrome (Alspaugh, M. A., Tan, E. M., 1975. J Clin Invest; 55 (5):1067-73) and is known by the alternative name SS-B (Sjögren syndromeantigen B).

With 2·10⁷ molecules per human cell it is an abundant protein which isfound in all tissues. La has a functional role in RNA metabolism and asa RNA chaperon, it processes pre-tRNA precursor molecules and influencesthe exactness as well as the efficiency of the RNA polymerase IIItranscription in vitro (inter alia Gottlieb, E. et al., 1989 EMBO J; 8(3): 841-50).

Chambers et al. (1988. J Biol. Chem., 263, 18043-18051) determined theamino acid sequence of La protein and 3 antigen epitope regions and madepredictions about the regions which are involved in RNA binding.

The primary structure of hLa protein (FIG. 1A; SEQ ID No. 1) can bedivided into three regions which form spatial domains that areindependent of each other (see FIG. 1B). The La motif is N-terminal,followed by central RNA recognition motif (RNA recognition motif, RRM)which is also referred to as RRM1. These two domains form the N-terminalhalf of the protein and together referred to as LaN. The second half,LaC, contains the C-terminal RRM (RRM2) as well as an adjoining long,flexible element of about 80 AS that exhibits no secondary structurecharacteristics. According to Chambers et al. (1988), the N-terminalRRM1 is particularly immunogenic.

McNeilage et al. (1984. J. Clin. Rennet. Immunol. 15, 1-17, Clin. exp.Immunol., 1985, 62, 685-695) examine anti La antibodies formed bypatients with autoimmune illnesses and which RNA and protein componentsare recognized by these antibodies. Scofield R H et al. examined thefine specificity of the autoimmune response against the Ro/SSA andLa/SSB ribonucleoproteins (1999, Arthritis Rheum. 42 (2):199-209).

Chan E K et al. (1987 J Exp Med 166 (6):1627-40) compare La epitopeswhich are recognized by human auto-antibodies and mouse antibodiesagainst human and bovine La. For this purpose, 5 monoclonal mouseantibodies were produced which were obtained by immunization of micewith bovine La. A cross-reactivity of the mouse antibodies with murineLa protein was not found.

Bachmann et al. (1990. Exp. Cell Res. 191, 171-180. 1991. Autoimmunity9, 99-107. 1992 Autoimmunity 12, 37-45) describe that the auto-antigenLa reaches the cell surface in UV-irradiated keranocytes and cellsinfected with herpes simplex type 1.

U.S. Pat. No. 5,457,029 describes a diagnostic test for detectinganti-La antibodies.

U.S. Pat. No. 4,751,181 discloses a procedure for producing the Laprotein antigen.

U.S. Pat. No. 4,784,942 discloses a murine anti-La antibody (hybridomaLA1, ATCC: HB-8609) which was obtained by immunization with bovine Laprotein.

Smith P R et al. (1985 J Immunol Methods 77(1):63-76) describe a murineanti-La antibody (SW5) which was obtained from rabbit by immunizationwith La protein.

Human monoclonal La antibodies were disclosed by Mamula M J (1989 JImmunol 143 (9):2923-8).

Offen D et al. (1990 J Autoimmune. 3 (6):701-13) describe murine anti-Laantibodies directed against an ideotype (16/6 Id) spread in SLEpatients.

In the following, the antibodies against the human La protein that areknown in the art are summarized:

-   -   La1B5 Bachmann et al. PNAS USA vol. 83: 7770-7774, 1986;    -   8G3 and 9A5 Mamula et al. J Immunol 143 (9):2923-2928, 1989;    -   anti-human La Carmo-Fonseca et al. ExpCellRes 185(1):73-85,        1989;    -   4B6 Tröster et al. J Autoimmunity 8 (6): 825-842, 1995;    -   SW1, SW3, SW5 Smith P R et al. J Immunol Methods. 77(1): 63-76,        1985; Pruijn et al. Eur J Biochem 232: 611-619, 1995;    -   3B9 Tran et al. Arthritis Rheum 46(1): 202-208, 2002;    -   DAB4 Al Ej eh et al. Nucl Med Biol. 2009, 36(4): 395-402.

All of these antibodies are monoclonal antibodies which bind to thehuman La protein. The CDR sequences of these antibodies are unknownexcept for SW5.

4B6 binds the La epitope with the sequence SKGRRFKGKGKGN (AS 330-343 ofthe human La protein, SEQ ID No. 2).

Al-Ej eh F. et al. (2007. Clin Cancer Res. 13 (18 Pt 2):55095-5518s)describe that the La protein is expressed at increased levels in tumorcells. It was found that, with increasing DNA damage, the anti-Laantibody 3B9 binds increasingly to tumor cells. It is described that theLa antigen is cross-linked in dead malignant cells by transglutaminase2. Based on this, Al-Ejeh F et al. (2007. Clin Cancer Res. 13 (18 Pt2):55195-5527s) describe in a mouse model the use of anti-La 3B9 for invivo targeting of tumor cells. Targeting was further improved byconcurrent cytostatic treatment. Al-Ejeh F et al. (2009. PLoS One. 4(2):e4630) describe a radio immunotherapy in which tumor targeting isdone with the monoclonal anti-La antibody DAB4. The radio immunotherapyis carried out in combination with chemotherapy. WO 2008/043148 A1discloses also combination therapy of anti-La antibodies with cytostaticagents.

In Al-Ejeh F et al. (2009. PLoS One. 2009; 4 (2):e4558) the use of theanti-La antibody DAB4 is described for the detection of the tumorresponse to DNA-damaging chemotherapeutic agents.

A general problem of the therapeutic effectiveness of monoclonalantibodies in tumor treatment is the binding capacity of the antibodiesto the cancer cells, i.e., the affinity of the antibodies and theselection of the suitable antigen which is bound by the antibodies.Specific tumor antigens mostly are not expressed in sufficient amountson the cancer cells. In cancer cells that sufficiently express tumorantigens, the binding rate of the used antibodies is often not highenough. Moreover, with a molecular mass of about 150 kDa antibodies arelimited in general with regard to tissue mobility. In this case,antibody fragments, like Fab, F(ab)₂ or scFv (single chain variablefragments), on account of their clearly smaller size, have considerableadvantages.

Bispecific antibodies, i.e., antibody derivatives of components of twodifferent monoclonal antibodies, offer new possibilities for therapyconcepts in cancer immunotherapy.

US 2005/0136050 A1 discloses bispecific antibodies which can bind to twodifferent targets. They serve for recruiting human immune effector cellsto a target antigen which is located on a target cell.

Quadromas are bispecific antibodies of the first generation and arecomprised of a heavy chain and a light chain of two different monoclonalantibodies. The two arms of the antibody are each directed againstdifferent antigens. The Fc part is formed jointly of both heavy chainsof the antibodies. With this construction it is, for example, possibleto position the paratope of an antibody directed against a tumor antigenand the paratope of a further antibody directed against a lymphocyteantigen onto one arm of the bispecific antibody, respectively. It is sopossible to form a three-cell complex resulting from the cells bound ineach case by the different paratopes and the effector cell bound by theFc part. In this context, an improved activation of the body's ownimmune cells arises generally relative to the tumor cells.

Bispecific antibodies of the newer generation are constructed of twodifferent scFv fragments. These are connected to each other by a linkerpeptide. Thus, a bispecific antibody can bind, for example, with onescFv to tumor cells and with the other scFv to effector cells.

When a paratope is directed against T cells, these cells can be alsoactivated. With normal monoclonal antibodies this is not possiblebecause T cells do not have Fc receptors. In addition, bispecificantibodies have a higher cytotoxic potential. They also bind to antigenswhich are expressed relatively weakly.

To this day, no bispecific antibodies have been approved for clinicaluse in humans.

Bispecific antibodies are known where an scFv binds to the CD3 complexon T cells, these are also called BiTE (bispecific T cell engager) (P.A. Baeuerle et. al., BiTE: Teaching antibodies to engage T cells forcancer therapy. Curr Opin Mol Ther 11, 2009, pages 22-30).

At the moment, two different BiTE antibodies are in clinical studies.Blinatumomab, an antibody directed against CD3 and CD19, is tested inpatients in late phases of Non-Hodgkin lymphoma and in patients withacute lymphoblastic leukemia of the B cell line (B-ALL). MT110 is anantibody which is directed against CD3 and EpCAM (epithelial celladhesion molecule) and is tested in patients with bronchial carcinomaand patients with gastrointestinal cancer diseases (R. Bargou en. al.,Tumor regression in cancer patients by very low doses of a Tcell-engaging antibody. Science 321, 2008, pages 974-977; K. Brischweinet al., MT110: a novel bispecific single chain antibody construct withhigh efficacy in eradicating established tumors. Mol Immunol 43, 2006,pages 1129-1143).

Not all monoclonal antibodies are suitable in the form of scFv fragmentsor for the construction of bispecific constructs. Particularly theaffinity of the antibodies is decisive which is determined by thevariable regions. Only particularly high-affinity antibodies are suitedas scFv fragments because binding occurs at the respective antigen onlywith one pair of variable regions of the heavy and light chains, incontrast to the complete IgG antibodies which have two pairs of variableregions of the heavy and light chains.

For the treatment of carcinomas there is a need for new therapeuticconcepts.

A big problem with immuno-targeting of cells, in particular inimmunotherapy of tumors, is either the absence of specific targets orthe loss of a specific target in some of the tumor cells. Therefore, nosuitable targeting module could be developed up to now for many targetcells.

The object of the invention is providing improved antibodies which binduniversal target structures on the surface of tumor cells.

SUMMARY OF THE INVENTION

The invention is based on the finding that the nuclear antigen La isreleased by injured or dying cells and can bind to neighboring intactcells where it is stable for more than 24 hrs. on their surface and isaccessible to the antibody. By antibody binding even NK cells can beactivated and thus cause an antibody-induced destruction (ADCC) of theintact cells.

In the context of tumor therapy (for example, with cytostatic agents orradiation therapy), the nuclear antigen La is released by apoptotictumor cells and binds to the surface of neighboring tumor cells.Therefore, the La protein represents an inducible universal surfacetarget (a universal target structure on the surface of the tumor cells).

The goal of the invention was therefore to develop antibodies orrecombinant antibody derivatives which allow to recognize target cells(in particular tumor cells) and to activate immune effector cells (e.g.,T cells, NK cells, dendritic cells) against them.

Another object of the invention is therefore providing new anti-Laantibodies, in particular with a high affinity to La which enable theuse of the antibodies as recombinant fragments for immuno-targeting.

The object is solved according to the invention by new anti-Laantibodies as well as recombinant antibodies which contain regionsdetermining complementarity (complementary determining regions, CDRs)which are characterized in that the CDRs of the variable region of thelight chain (V_(L)—left column) and the CDRs of the variable region ofthe heavy chain (V_(H)—right column) comprise the sequences disclosed inone of the Tables 1 to 8:

TABLE 1 Antibody 5B9: SEQ ID SEQ V_(L) No. V_(H) ID No. CDR1KSSQSLLNSRTPKNYLA 3 HYYIY 4 CDR2 WASTRKS 5 GVNPSNGGTHFNEKFKS 6 CDR3KQSYNLLT 7 SEYDYGLGFAY 8

TABLE 2 Antibody 7B6: SEQ SEQ ID ID V_(L) No. V_(H) No. CDR1RSSQSLLDSRTRKNYLA 9 DFWMN 10 CDR2 WASTRES 11 QIRNKPNNYETYYSDSLKG 12 CDR3KQSYNLPT 13 LGNSWFAY 14

TABLE 3 Antibody 22A: SEQ SEQ V_(L) ID No. V_(H) ID No. CDR1 SASSSVSYMY15 NYYIY 16 CDR2 DTSKLAS 17 YIYPGNGGTAYNQKFKD 18 CDR3 QQWSSNPQ 19RGALGYYFDY 20

TABLE 4 Antibody 24BG7: SEQ SEQ V_(L) ID No. V_(H) ID No. CDR1SASSSVTYMEI 21 NYGIS 22 CDR2 DTSKLAS 23 EIYRGSGNSYYNEKFKG 24 CDR3QQWISNPPT 25 GGLSFAY 26

TABLE 5 Antibody 312B: SEQ SEQ V_(L) ID No. V_(H) ID No. CDR1RASENIYTYLA 27 DYWIE 28 CDR2 NAKTLAE 29 EILPGSVSIKYNEKFKG 30 CDR3QHHYGTPYT 31 SRSIYDGYFYY 32

TABLE 6 Antibody 27E: SEQ SEQ V_(L) ID No. V_(H) ID No. CDR1 SASSSVSYMY33 SYGIN 34 CDR2 RTSNLAS 35 EIYPGSGTTFYNEKFRG 36 CDR3 QQYHSYPRT 37HGGYPFYFDY 38

TABLE 7 Antibody 2F9: SEQ SEQ V_(L) ID No. V_(H) ID No. CDR1RASESVDSYGNSFMH 39 TSGMGVS 40 CDR2 RASNLES 41 HIYWDDDKGYNPSLKS 42 CDR3QQSNEDPPT 43 GDVEFDY 44

TABLE 8 Antibody 32A SEQ SEQ V_(L) ID No. V_(H) ID No. CDR1 SASSSVSYMY45 TYGLT 46 CDR2 RTSNLAS 47 EIFPGSGTTFYNEKFND 48 CDR3 QQYHSYPRT 49YSNYPYYFDY 50

The antibodies comprise preferably the following, optionally humanized,variable regions of the light chains (V_(L)) and/or heavy chains(V_(H)):

TABLE 9 Antibody V_(L) SEQ ID No. V_(H) SEQ ID No. 5B9 51 52 7B6 53 5422A 55 56 24BG7 57 58 312B 59 60 27E 61 62 2F9 63 64 32A 65 66

The anti-La antibody according to the invention, herein also referred toas “anti-La”, are characterized by their CDRs and preferably theaforementioned amino acid sequences of the variable regions of the heavyand the light chains.

Subject matter of the invention are also gene sequences which code forthe aforementioned amino acid sequences of the variable regions of thelight chains (V_(L)) and/or heavy chains on (V_(H)):

TABLE 10 Antibody V_(L)-DNA SEQ ID No. V_(H)-DNA SEQ ID No. 5B9 67 687B6 69 70 22A 71 72 24BG7 73 74 312B 75 76 27E 77 76 2F9 79 80 32A 81 82

Advantageously, the anti-La antibodies according to the invention canstill bind the La epitope even when the nuclear antigen is present boundto the surface of cells. In a cell culture model it has beendemonstrated that the anti-La antibodies according to the invention bindto La protein bound to the cell surface and the cells are killed by NKcells in an antibody-dependent cell-induced cytotoxicity (anti-bodydependent cellular cytotoxicity—ADCC).

The epitopes recognized by the antibodies according to the invention onthe human La protein are summarized in the following Table 11:

TABLE 11 Redox Antibody Structure of the epitope epitopes (AS of hLa)dependency 5B9 linear, SEQ ID No. 83  95-104 (−) 7B6 linear, SEQ ID No.84 311-328 (+) oxidized 22A conformation 107-200 (−) 24BG7 conformation107-200 (−) 312B conformation  10-100 (+) reduced 27E conformation 10-100 (+) reduced 2F9 conformation  10-100 (+) reduced 32Aconformation  10-100 (+) reduced

Particularly preferred is the antibody 7B6 or an antibody with theaforementioned CDRs of the variable regions of the light chains (V_(L))and heavy chains (V_(H)) (SEQ ID Nos. 9 to 14) and preferably thevariable regions of the light chains (V_(L)) and/or heavy chains (VH)according to SEQ ID Nos. 53 and 54 or corresponding humanizedstructures. Advantageously, this antibody binds a redox-dependentepitope which becomes accessible under oxidative conditions in the Laprotein. Such oxidative conditions (“oxidative stress”) exist in tumortissue, in particular as a reaction to radiation and/or chemotherapy (inparticular cytostatic agent therapy). Oxidative stress refers to ametabolism situation in which reactive oxygen compounds (ROS—reactiveoxygen species) are formed. The fact that the 7B6 epitope under theseoxidative conditions becomes accessible in La enables a more specificanti-tumor therapy with reduced side effects. La which is released inother tissue (non-tumor tissue) by dying cells under non-oxidativeconditions is not bound by this antibody. The epitope of 7B6 is madeaccessible only by oxidative stress. Otherwise, the epitope is crypticand the antibody cannot bind to the La protein.

Further preferred anti-La antibodies are 312B, 27E, 2F9, 32A orantibodies with the CDRs of the variable regions of the light chains(V_(L)) and/or heavy chains (V_(H)) selected from the SEQ ID Nos. 27 to50 (preferably in the combinations set forth in the Tables 5 to 8).These bind to split, conformation-dependent epitopes in the human Laprotein which comprise the areas of the amino acid sequences of aminoacid 10-20 and amino acid 94-100 (in the above inserted Table and in thefollowing also “10-100”) of the human La protein. The inventors havefound that these epitopes are sensitive to oxidation.

These anti-La antibodies recognize exclusively the reduced form (i.e.native form) of the La protein and not the oxidized form. Because insome tumors hypoxic conditions are present, these anti-La antibodies areparticularly suitable for such hypoxic tumors, in particular when ananti-La targeting is desired without combination with therapies (inparticular chemotherapy and/or radiation therapy) which cause oxygenstress and thus an oxidation of the La protein. Hypoxic conditions areto be understood as oxygen depletion (i.e. an oxygen concentration whichis (significantly) lower than that in healthy tissue). This oxygendepletion often originates in solid tumors, in particular when the tumor(or parts thereof) is growing faster than the blood-supplying tissue.This is a special advantage because hypoxic tumors are often resistantagainst chemotherapy and radiation therapy.

Anti-La antibodies that are also preferred are 22A and 24BG7 orantibodies with the CDRs of the variable regions of the light chains(V_(L)) and/or heavy chains (V_(H)) selected from the SEQ ID Nos. 15 to26 (preferably in the combinations set forth in Tables 3 and 4). Thesebind to split epitopes in the human La protein which comprise theregions of the amino acid sequences of amino acid 107-116 and amino acid185-200 (in the following also “107-200”) of the human La protein.

The antibodies according to the invention were obtained by differentimmunization methods (see FIG. 2). In healthy individuals noimmunological reactions take place against the body's own proteinsbecause auto-reactive T cells and B cells are eliminated during theirdevelopment. This tolerance and the homology between hLa and mLa may bethe reason that mice hardly developed antibodies against rhLa proteinafter conventional immunization with recombinant human La protein(rhLa). After a plurality of fusions (greater than 50) only the two mAks5B9 and 7B6 were obtained. Instead, a strong B cell response against thehLa antigen in hLa transgenic mice was induced by adoptive T celltransfer and several mAks could be obtained therefore from one mouse.

The isotypes of the antibodies according to the invention and theimmunization methods for their production are summarized in thefollowing Table 12:

TABLE 12 Antibody Isotype Preparation 7B6 IgG1 immunization with rhLaprotein 5B9 IgG2a immunization with rhLa₁₋₁₉₂ 24BG7 IgG1 immunization ofmice transgenic for human La protein (hLaTg), adoptive T cell transfer22A IgG1 hLaTg, adoptive T cell transfer 27E IgG2b hLaTg, adoptive Tcell transfer 312B IgG1 hLaTg, adoptive T cell transfer 2F9 IgG1 hLaTg,adoptive T cell transfer 32A IgG1 hLaTg, adoptive T cell transfer

All antibodies according to the invention are capable of recognizingrecombinant human La protein (rhLa, independent of whetherprokaryote-produced or eukaryote-produced) and native human La proteinin immunoblot (western blot). The antibodies 22A and 32A areadvantageously specific for the human La protein and cannot bind therelated murine La protein at all. mAk 7B6 differentiates betweenprokaryote-produced and eukaryote-produced murine La proteins (mLa) andrecognizes only the bacterial, probably not post-translationallymodified, variant of the murine La protein (see FIG. 4). The mAks 22A,27E and 312B show the strongest binding to human La protein inimmunoprecipitations. 312B was suited best for the precipitation of themLa protein (FIG. 5).

In the following Table 13, the reactivities of the antibodies accordingto the invention in immunoblot, in immunoprecipitation and immunefluorescence are summarized:

TABLE 13 Immuno- Reactivity fluorescence after Reactivity in againstImmuno- para-formaldehyde immunoblot against 3T3 transgenicprecipitation (PFA) fixation human murine for hLa in from 3T3 LaG humanmurine cells cells immunoblot total extract cells cells mAk rhLa rmLa(HeLa) (3T3) hLa mLa hLa mLa (HeLa) (3T3) 7B6 +++ ++ +++ − ++ − − − (+)− 5B9 +++ +++ +++ +++ +++ +++ − − ++ − 24BG7 +++ + +++ + ++ + + − ++ ++22A +++ − +++ − + − ++ − ++ − 27E +++ + +++ +++ ++ ++ + + +++ +++ 312B+++ +++ +++ +++ + ++ +++ +++ +++ +++ 2F9 +++ +++ ++ ++ + + − − + + 32A+++ − ++ − (+) − − − (+) − 3T3 LaG: murine cells transgenic for hLa. Theevaluation of the binding strengths was carried out according to thefollowing scale: − negative, +++ strongly positive, ++ positive, +weakly positive, (+) very weakly positive.

The affinities of the native antibodies are within the magnitude of10⁻¹⁰ mol/l. The affinities of the recombinant derivatives reach 10⁻⁷ to10⁻⁹ mol/l. Due to the high affinity, the CDR sequences according to theinvention are suitable in particular for the production of recombinantfragments (like scFv) and for immunotargeting.

The term “antibody” in the meaning according to the inventionencompasses all antibodies, antibody fragments and derivatives thereofwhich are able to bind to the antigen, in this case the human Laprotein, and comprise the CDRs according to the invention completely orpartially. They include the complete monoclonal antibodies and also theepitope-binding fragments of these antibodies. In this context, theepitope-binding fragments (here also referred to as antibody fragmentsor antibody derivatives) comprise all parts of the antibody which areable to bind to the antigen, in this case the human La protein. Examplesof preferred antibody fragments according to the invention include, butare expressly not limited to, Fab, Fab′, F(ab′)₂, Fd, individual chain(single chain) variable fragments (scFv), single chain antibodies,disulfide-linked variable fragments (sdFv), and fragments that containeither a variable region of the light chain (V_(L)) or a variable regionof the heavy chain (V_(H)). Furthermore, included are recombinantantibodies, like diabodies, triabodies and tetrabodies.

Preferably, the antibody carries a marker molecule, as for examplebiotin, dioxygenin, a radionuclide or a fluorescent dye. It isparticularly preferred that the antibody is conjugated with an effectorgroup.

Antibody fragments contain the variable regions either alone or incombination with other regions which are selected from the hinge region,and the first, second and third segment of the constant region (C_(H)1,C_(H)2, C_(H)3). Also encompassed by the term “antibody” are chimericantibodies where different parts of the antibody originate fromdifferent species, as for example antibodies with a murine variableregion which is combined with a human constant region.

Antibody fragments are linked optionally with each other by a linkerpeptide. The linker peptide comprises a short (preferably having alength of 10 to 20 amino acid residues), flexible peptide sequence whichis selected such that the antibody fragment has such a three-dimensionalfolding of the V_(L) and V_(H) that it exhibits the antigen specificityof the complete antibody. Preferred linker peptides are glycine-serinelinkers with the structure (Gly_(X)Ser)_(Y) with x and y selected from 1to 10, preferably 3 to 5. Furthermore, linker peptides are preferredwhich are comprised of a peptide sequence which can increase theprotease resistance of the antibody derivatives. Particularly preferredare linker peptides according to SEQ ID No. 85 (E7B6).

The term “variable region” is defined according to the invention as theparts of the heavy and light chains of the antibodies which differbetween antibodies in their sequence and determine the specificity ofthe antibody and the binding action to its antigen. In this context, thevariability is not distributed evenly in the variable region. It isusually concentrated within three defined segments of the variableregion which are referred to as complementarity determining region(CDRs) or also hypervariable regions and exist in the variable regionsof the light as well as the heavy chains. The more strongly preservedparts of the variable regions are called frame regions (frameworkregions). The variable regions of the heavy and light chains containfour framework regions which adopt predominantly a beta sheet structure,wherein every framework region is connected with three CDRs which formloops which connect the beta sheet structures and in some cases form apart of the beta sheet structure. The CDRs of the respective chain arebrought into immediate proximity by the framework regions and contributetogether with the CDRs of the other chain to the formation of theantigen binding region of the antibodies.

The constant region (Fc) of the antibodies is not involved in antigenbinding but offers, instead, varied effector functions which aretriggered by binding to the appropriate Fc binding receptors, as forexample the induction of the antibody-dependent cellular cytotoxicity(ADCC).

Preferably, the antibodies according to the invention comprise at leastone variable region of the heavy chain (V_(H)) and a variable region ofthe light chain (V_(L)) in form of an scFv. In this context, thevariable region of the heavy chain (V_(H)) and the variable region ofthe light chain (V_(L)) each contain at least one of the CDR sequencesaccording to the invention.

In the antibodies according to the invention certain amino acids of thespecific amino acid sequences can be exchanged in such a way that theymaintain the binding properties of the anti-La antibody, but differ intheir sequence by exchange, deletion or addition of one or several aminoacids. Encompassed are therefore also antibodies which containstructures whose amino acid sequences, compared to the amino acidsequences according to the invention of the variable regions selectedfrom the sequences according to SEQ ID Nos. 51 to 66, have a sequenceidentity of preferably at least 70%, particularly preferred at least80%, in particular at least 90%, or contain appropriate humanizedsequences and that bind the antigen La, wherein these sequences comprisesix of the CDRs according to SEQ ID Nos. 3 to 50 in combinations thatare presented in the Tables 1 to 8.

In special embodiments of the invention, the antibody comprises inaddition to the amino acid sequences according to the invention of thevariable regions or appropriate humanized sequences, the followingstructures:

-   -   a constant region of a heavy chain of a human IgG,    -   a region C_(L) of the human kappa light chain    -   and a human IgG hinge region,        optionally in the form of a F(ab)₂-fragment.

In a particularly preferred embodiment of the invention, antibodies inthe form of scFv fragments which comprise at least one variable regionof the heavy chain (V_(H)) and/or a variable region of the light chain(V_(L)) which contain CDR regions according to the invention.Furthermore, particularly preferred are antibodies in the form of scFvfragments which comprises at least one of the variable regions accordingto the invention of the heavy and/or light chain.

The invention encompasses murine anti-La antibody and humanized versionsof these antibodies.

The goal of the humanization of antibodies resides in the reduction ofthe immunogenitity of a xenogenic antibody, like in this case of themurine antibody, for the use in the human system, wherein the fullbinding affinity and the antigen specificity is preserved. Humanizedantibodies can be produced in different ways, as for example byresurfacing and CDR grafting. In resurfacing, all non-CDR regions on thesurface of the antibody are changed by a combination of molecularmodeling, statistical analyses and mutagenesis so that they resemble thesurface of antibodies of the target organism. In CDR grafting, the CDRregions according to the invention are introduced into human variableregions.

Humanized antibodies which contain the CDR regions according to theinvention are expressly considered to be components of the invention.

Components of the invention are also antibodies which are conjugatedwith an effector group. Conjugation is to be understood here as couplingof a substance to an antibody. Coupling of the antibody with theeffector group is produced preferably by expression as a fusion proteinor by in vitro methods wherein the effector group is preferably coupledby linker groups to the antibody (for example, by thioether bonds ordisulfide bonds). They can be bound also to the antibody by anintermediary carrier molecule, for example, serum albumin. Optionally,an antibody also contains several effector groups.

In this context, the effector groups are preferably pharmaceuticallyeffective substances (active substances). Preferred active substancescomprise, but are not limited to, toxins, like cytostatic agents, forexample, maytansinoids and maytansinoid analogues, taxoids, CC-1065 andCC-1065 analogues, dolastatin and dolastatin analogues, methotrexat,daunorubicin, doxorubicin, vincristin, vinblastin, melphalan, mitomycinC, chlorambucil and calicheamicin. The invention also encompassesantibodies which are conjugated with radionuclides as effector groupsand their use for the therapy and diagnostics, in particular of tumors.Suitable radionuclides are preferably the radioactive isotopes oftechnetium, rhenium, yttrium, copper, gallium, indium, bismuth andplatinum, as for example ^(99m)Tc, ⁹⁰Y, ¹⁸⁶Rc, ¹⁸⁸Rc, ⁶⁸Ga and ¹¹¹In.

Effector groups according to the invention comprise furthermore enzymes(particularly enzymes that are suitable for the ADEPT system),co-stimulatory molecules (e.g., CpG) or also nucleic acids. The antibodythat is conjugated with an effector group can be present optionally inthe form of a fusion protein.

Subject matter of the invention are also recombinant antibodies,containing:

-   -   i. a binding unit of an antibody that binds specifically to an        epitope of a human nuclear antigen, preferably human La protein,        as well as    -   ii. a binding unit        -   of an antibody which binds specifically to an effector cell            or        -   of a ligand which binds specifically to an effector cell.

The antibody or ligand which binds specifically to an effector cell isselected preferably from antibodies and ligands which bind specificallysurface structures on T lymphocytes, NK cells, dendritic cells,granulocytes and/or monocytes.

The epitope, which the antibody which binds specifically to an epitopeof a human nuclear antigen, is accessible preferably only underoxidizing conditions. Such a preferred antigen which is recognized byanti-La 7B6 is indicated in SEQ ID No. 84.

Components of the invention are therefore also recombinant antibodieswhich are conjugated with a further antibody, antibody fragment orligand which is directed against an antigen that is different from thehuman nuclear antigen, in particular the human La protein. Therecombinant antibody is preferably a bispecific antibody. The bispecificantibody is preferably a single chain bispecific diabody (scBsDb). Inthis case, two scFv fragments are connected to each other by a shortlinker (preferably of a length of 10 to 20 amino acid residues).Particularly preferred, the bispecific antibody is a single chainbispecific tandem antibody (scBsTaFv). In this case the two scFvfragments are connected by longer linker peptides (preferably of alength of from 18 to 50 amino acid residues) which results in anespecially flexible structure.

Preferably, the recombinant antibodies according to the inventioncontain, in addition to the La antibody according to the invention, anantibody, antibody fragment or ligand which is directed specificallyagainst surface antigens of effector cells, as for example T cells,particularly cytotoxic T cells, NK cells, monocytes, macrophages,dendritic cells, or granulocytes. The definition of effector cells inaccordance with the invention encompasses all the cells of the innateand adaptive immune system which provide immunological reactions or areinvolved actively therein. These antibodies are particularly preferablydirected against the following surface structures on effector cells:CD3, CD8, CD4, CD25, CD28, CD16, NKG2D, NKp46, NKp44, activating KIRreceptors (activating killer cell immunoglobulin-like receptors).

Also components of the invention are recombinant antibodies whichcomprise in addition to the La antibody according to the invention aligand which influences the activity of effector cells by binding to thesurface of the effector cells. The ligand is selected in this contextsuch that it binds specifically to surface structures of effector cellsand triggers upon binding signal cascades for the activation of theeffector cells. Preferred as a ligand is a protein structure or a glycanwhich binds specifically to a receptor which is expressed specificallyon the surface of effector cells, wherein the ligand causes anactivation of the effector cell by its binding to the receptor.Particularly preferred, the protein structures are selected from ULB-Ps(e.g., ULB-P2), MICA, MICB, as well as cytokines (as for example IL2 andIL15) and their fusion proteins.

Binding of the anti-La antibody according to the invention to thefurther antibody, antibody fragment or to the protein structure isproduced preferably by an expression as a fusion protein or by in vitromethods, wherein the further antibodies, antibodies or proteinstructures are bound preferably by linkers, like peptide linkers, to theantibody.

The invention comprises furthermore nucleic acid sequences which encodefor an antibody according to the invention, as well as vectors whichcontain the nucleic acid sequences.

The vector (expression vector) is in each case preferably a plasmid, anartificial chromosome or even a virus particle or another vector whichcontains an expression cassette which is incorporated stably in thegenome of the host (host cell or host organism).

Preferably, the nucleic acid sequences which code for an antibodyaccording to the invention contain the sequences which code for the CDRsmentioned above (selected from SEQ ID Nos. 3 to 50 in the combinationsset forth in Tables 1 to 8) of the variable regions of the heavy chains(V_(H)) and the light chains (V_(L)).

In another preferred embodiment of the invention, the nucleic acidsequences contain sequences which code for the variable regions of theheavy chains (V_(H)) and/or the light chains (V_(L)) selected from SEQID Nos. 51 to 66.

Components of the invention are also host cells or non-human hostorganisms which contain a nucleic acid sequence according to theinvention.

A host cell is a naturally occurring cell or a transformed orgenetically modified cell line or a (multicellular) non-human hostorganism which contains the expression system according to the invention(i.e. at least one expression vector). In this context, the inventioncomprises transient transfectants (e.g., by mRNA injection), i.e., hosts(host cells or host organisms) in which the expression system iscontained as a plasmid or artificial chromosome, as well as hosts inwhich the expression system is integrated stably into the genome of thehost (or single cells of the host). The host cell is selected preferablyfrom prokaryotes or eukaryotes. Preferred prokaryotes are selected fromEscherichia coli and Bacillus subtilis. Preferred eukaryotes are yeastcells (e.g., Saccharomyces cerevisiae, Pichia pastoris), insect cells,amphibian cells or mammalian cells, as for example CHO, HeLa, Hek293T,Hek293A. Preferred host organisms are plants, as for example maize ortobacco, invertebrates or vertebrates, in particular Bovidae, Drosophilamelanogaster, Caenorhabditis elegans, Xenopus laevis, Medaka, zebra fishor Mus musculus, or cells or embryos of the aforementioned organisms.

The invention encompasses furthermore a pharmaceutical composition whichcontains one or several antibodies according to the invention inassociation with a pharmaceutical suitable diluent or carrier.Preferably, the pharmaceutical composition is present in a form suitablefor intravenous administration.

Preferably, the composition comprises a chimeric or humanized antibodywith a reduced immunogenicity which contains the CDR regions accordingto the invention.

The pharmaceutical compositions according to the invention comprisedifferent dosage forms. The pharmaceutical compositions are administeredpreferably parenterally, particularly preferred intravenously. In oneembodiment of the invention, the parenteral pharmaceutical compositionis present in an administration form which is suitable for injection.Particularly preferred compositions are therefore solutions, emulsionsor suspensions of the antibody which is present in a pharmaceuticalsuitable diluent or carrier.

As carriers, preferably water, buffered water, 0.4% saline solution,0.3% glycine and similar solvents are used. The solutions are sterile.The pharmaceutical compositions are sterilized by customary, well knowntechnologies. The compositions contain preferably pharmaceuticalacceptable excipients, as for example those that are required to provideapproximately physiological conditions and/or to increase the stabilityof the antibody, as for example agents to adjust the pH value andbuffering agents, agents for adjusting the toxicity and the like,preferably selected from sodium acetate, sodium chloride, potassiumchloride, calcium chloride and sodium lactate. The concentrations of theantibodies according to the invention in these formulations are variabledepending on the application; they amount preferably to less than 0.01%by weight, preferably at least 0.1% by weight, further preferred between1 and 5% by weight, and they are selected primarily on the basis offluid volume, viscosity, and the like or in accordance with therespective mode of administration.

The antibodies according to the invention are preferably taken up in acomposition which is suitable for parenteral administration. Preferably,the pharmaceutical composition is an injectable buffered solution whichcontains between 0.1 to 500 mg/ml of antibody, particularly preferredbetween 0.1 to 250 mg/ml of antibody, in particular together with 1 to500 mmol/l (mM) of a buffer. The injectable solution can be present in aliquid as well as a lyophilized dosage form. The buffer can bepreferably histidine (preferably 1 to 50 mM, particularly preferred 5 to10 mM) at a pH value of from 5.0 to 7.0 (particularly preferred at a pHvalue of 6.0).

Other suitable buffers comprise, but are expressly not limited to,sodium succinate, sodium citrate, sodium phosphate or potassiumphosphate. Sodium chloride is preferably used between 0 to 300 mM,particularly preferred 150 mM for a liquid administration form. For alyophilized administration form the pharmaceutical composition containspreferably an antifreeze agent, preferably 0-10% sucrose (particularlypreferred 0.5-1.0%). Other antifreeze agents encompass trehalose andlactose. For a lyophilized administration form the pharmaceuticalcomposition contains preferably expanding agents, preferably 1 to 10%mannitol. Other expanding agents comprise glycine and arginine. Inliquid as well as in lyophilized administration forms stabilizers arepreferably used, particularly preferred between 1 to 50 mM ofL-methionine (particularly preferably between 5 and 10 mM).

In a preferred embodiment, the pharmaceutical composition comprises theantibody in a dosage amount from 0.1 mg/kg to 10 mg/kg peradministration. Particularly preferred dosage amounts comprise 1 mg/kgof body weight.

Pharmaceutical compositions must be sterile and stable under theproduction and storage conditions. The composition can be formulated asa solution, microemulsion, dispersion, in liposomes or another orderedstructure which is suitable for high concentrations of antibody. Sterileinjectable solutions can be produced in that the necessary amount of theantibody is taken up in a suitable solvent, with one or with acombination of the above enumerated ingredients as needed, followed byfiltration sterilization. For sterile, lyophilized powders for preparingsterile injectable solutions the preferred preparation procedures arevacuum drying and spray drying which results in a powder of the antibodyplus any additional desired ingredients from a solution thereof that hasbeen sterile-filtered beforehand.

The invention encompasses the use of the antibody according to theinvention as a medicament.

The invention also encompasses a method for treating a human beinghaving a tumor disease by administering an antibody according to theinvention.

Tumor diseases mean in particular acute myeloid leukemia (AML), chronicmyeloid leukemia (CML) and pro-myeloid leukemia (PML) and otherillnesses such as myelodysplastic syndrome (MDS). In addition tohematologic tumors, the anti-La antibodies according to the inventionare suited in particular for the therapy of solid tumors, e.g., of theprostate, pancreas, colon, lung, mamma cancer, cancer of the thyroid,melanoma, brain tumors (e.g., glioblastomas) as well as head and necklymphomas.

Advantageously, the invention is suited in particular for immunotherapyof so-called “immune escape” variants of tumors, i.e. tumors which havestopped or down-regulated the expression of tumor antigens in order toescape the immune system. According to the invention, no antibody isused for tumor targeting which is directed against a tumor antigen butagainst a nuclear antigen. By the recombinant antibodies according tothe invention which bind the La protein (or another nuclear antigen) onthe surface of tumor cells as well as effector cells and thus recruitthem to the tumor cells, tumor cells can thus be targeted with anantigen on the surface which is expressed in all cells as a nuclearantigen constitutively and is not tumor-specific.

Particularly preferred for an immunotherapy is the antibody 7B6 or anantibody with the aforementioned CDRs of the variable regions of thelight chains (V_(L)) and heavy chains (V_(H)) (SEQ ID Nos. 9 to 14) andpreferably the variable regions of the light chains (V_(L)) and/or heavychains (V_(H)) according to SEQ ID Nos. 53 and 54 or correspondinghumanized structures. As already discussed above, this antibody binds aredox-dependent epitope, which is released under the conditions of“oxidative stress” as it is particularly existing in tumor tissue. Thefact that the 7B6 epitope under these oxidative conditions becomesaccessible in La enables a more specific anti-tumor therapy with reducedside effects.

For therapeutic uses a sterile pharmaceutical composition, containing apharmacologically effective dosage amount of one or several antibodiesaccording to the invention, is administered to a patient in order totreat the aforementioned illnesses.

Killing of the tumor cells is achieved preferably by the recruitment ofeffector cells. As an alternative, or in addition, by the specifictransport of pharmacological active substances (e.g., toxins) and therelease thereat.

In a special embodiment of the invention, an antibody is used in theform of a bispecific antibody. In this context, the bispecific antibodyin a particularly preferred embodiment of the invention contains atleast a binding unit of an antibody that specifically binds to anepitope of a human nuclear antigen, preferably human La, as well as abinding unit which is directed against a surface structure on NK cells,preferably against ULB-P2 (i.e. preferably a binding unit of ananti-ULB-P2 or of the ULB-P2 ligand). Preferably, the bispecificantibody is used for the treatment of AML, as well as prostate cancer.

In a further especially preferred embodiment of the invention, thebispecific antibody contains at least a binding unit of an antibody thatbinds specifically to an epitope of a human nuclear antigen, preferablyhuman La, as well as a binding unit which is directed against a surfacestructure on T cells, preferably against CD3 or CD8. Preferably, thebispecific antibody is used for the treatment by AML as well as prostatecancer.

In both aforementioned preferred embodiments of the invention thebinding unit of the antibody which binds specifically human La containspreferably 6 CDRs according to SEQ ID Nos. 3 to 50 in a combinationpresented in the Tables 1 to 8.

In addition to the application in medicine for therapeutic purposes, theantibodies according to the invention are suited for diagnostics,biological research and other applications in which the detection of theLa protein is of interest. Such uses are in particular western blot,immune staining of cells (e.g., for flow cytometry and microscopy) andELISA, as well as the use as a tracer in imaging technologies as forexample CT (computer tomography), PET/CT (positron emission tomography).

The invention also encompasses a method for producing an antibody withthe steps:

-   -   a. immunization of a first recipient with antigen wherein the        antigen is a foreign antigen for the first recipient,    -   b. isolation of T lymphocytes from the immunized recipient,    -   c. transfer of the isolated T lymphocytes into a second        recipient which expresses the antigen, i.e. the antigen is an        auto-antigen for the second recipient,    -   d. isolation of antibody-producing B cells from the second        recipient.

Step a. corresponds to a classical immunization. The immunization can becarried out with or without adjuvant, e.g., with peptides or recombinantprotein.

The first and second recipients are selected preferably from non-humanvertebrates or non-human mammals, in particular rodents, like mice,hamsters or rats.

The antigen is for the first recipient a foreign antigen, i.e. the firstrecipient does not express the antigen.

Between steps b. and c. an enrichment of CD4 cells is preferably carriedout.

The second recipient in step c. expresses the antigen, preferablyconstitutively, particularly preferred as a transgene. This means forthe second recipient that the antigen is an auto-antigen. With theexception of the expressed antigen the second recipient is otherwisegenetically identical to the first recipient as much as possible.

After step d. a selection of antigen-specific B cells, hybridomageneration and screening of generated hybridomas, and antibodyproduction according to conventional methods, or the production andscreening of recombinant antibody libraries, is carried out.

Preferably, the variable regions of the light chain (V_(L)) and thevariable region of the heavy chain (V_(H)) of the antigen-recognizing Bcells are sequenced and the CDRs are determined. Based on these sequencedata, recombinant antibody fragments and/or humanized antibodies can beproduced advantageously.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures and embodiments explained the invention in moredetail without limiting the invention to them. The data of themonoclonal antibody SW5 are given as a comparative example.

FIG. 1A shows the amino acid sequence of the human La protein (SEQ IDNo. 1). In the sequence the epitopes which are recognized by antibodies(SW5, 3B9 as well as 4B6) known in the prior art are underlined twice.The epitopes that are recognized by the antibodies 5B9 and 7B6 accordingto the invention are marked by black bars. The two spatial epitopes (ACE10-100 or 107-200) and the matching anti-La mAk (312B, 27E, 2F9, 32A, or22A, 24BG7) are also indicated. It should be emphasized that adeletion/mutation of the first or last five amino acids (black widerbar) causes loss of reactivity.

FIG. 1B shows a schematic illustration of the hLa protein with its threedomains and functional areas. The different epitope regions are shownand the names of the mAks which bind them are indicated.

FIGS. 2A-2B explain the different technologies for the generation of thedifferent anti-La-mAks. FIG. 2A) The mAks 7B6 and 5B9 were obtained fromwild type (wt) mice which had been immunized either with recombinanthuman La protein (rhLa) or with rhLa1-192 antigens. FIG. 2B) Theremaining mAks come from one single mouse. The latter was transgenic forhLa (hLaTg) and received T cells from a wt mouse which had beenimmunized before with rhLa.

FIG. 3 shows the detection of the hLa-transgene in some of the hybridomacells.

Total extracts were produced from the different hybridoma cells andapplied onto a 12% SDS gel. After electrophoresis and western blot on aPVDF membrane, hLa and mLa proteins were detected by anti-La-5B9 andanti-mouse-IgG-AP (AP=alkaline phosphatase). The secondary antibody(sek.) alone showed no protein band.

FIG. 4 shows the reactivity of the different mAks according to theinvention against human and murine La protein by western blot(immunoblot). Bacterially produced recombinant human or murine Laprotein (rhLa or rmLa) as well as total extracts by human HeLa cells(DSMZ ACC 57) or by murine 3T3 cells (ATCC CRL 1658) were applied tofour separate 12% polyacrylamide gels. After western blot each membranewas cut into strips which were incubated with the different hybridomasupernatents. The detection was done with anti-mouse-IgG-AP.

All antibodies recognized rhLa and eukaryotic hLa protein. The mAks 22Aand 32A showed no reactivity at all towards rmLa and mLa, while 5B9,24BG7, 2F9, 27E and 312B could recognize hLa as well as mLa. 7B6 and SW5exhibited the special effect that they could stain bacterially producedrmLa but not eukaryotic mLa in immunoblot. The reason could be apost-translational modification of the protein in the respective epitoperegions which is present in 3T3 cells but not in bacteria.

FIG. 5 shows immunoprecipitation (IP) of hLa and mLa with the differentanti-La-mAks.

After precipitation of the immunocomplexes by TrueBlot™ anti-mouse Ig IPbeads and addition of SDS sample buffer, the samples were applied to a12% polyacrylamide gel. SDS PAGE was followed by an electrophoretictransfer of the proteins onto a nitrocellulose membrane. After blockingof the membrane, the La proteins were detected with 5B9 hybridomasupernatent and mouse IgG TrueBlot™-POD. 3T3 LaG total extract (TE)served as a positive control. The different running behavior of hLa andmLa is recognizable as a double band. As a negative control (NK), noanti-La-mAk was added to evaluate the background resulting from thebeads. Parallel to this, the reactivity was checked for every mAkindividually against 3T3 LaG total extract by immunoblot. The detectionwas done with anti-mouse-IgG-AP.

The recognition and stable binding of the native antigen can be verifiedby immunoprecipitation. The La-anti-La immunocomplexes can be isolatedby TrueBlot™ anti-mouse Ig IP beads and afterwards the La antigen can bedetected by immunoblot. As a test system a total extract of a mouse-cellline which was stably transfected with hLa (3T3 Lying) was used. In thisway, it was possible to test binding to both proteins at the same time.In addition, the total extract of these cells was applied to another SDSgel to check in parallel the reactivity of the hybridoma supernatentsagainst both proteins by immunoblot.

FIGS. 6 to 8 show the characterization of the peptide epitope recognizedby the anti-La antibody 7B6.

For this purpose, a series of deletion mutants of the La protein wasproduced (FIG. 6 and FIG. 7). The purified proteins were analyzed bywestern blot (FIG. 8) with anti-penta HIS antibody (C (I)) and theanti-La mAk 7B6 (C (II)). In the left column of the FIGS. 6 and 7 themutants are shown, respectively. The right column shows the reactivityin western blot. The results show that the antibody 7B6 recognizes theepitope with AA311 to 328 (SEQ ID No. 84).

FIGS. 9A-9C explain schematically the mechanism of action of the tumortherapy with the anti La antibodies according to the invention.

FIG. 9A: Cellular stress, e.g., by medicament (cytostatic agents, radiopharmaceuticals), radiotherapy or TAA-specific immuno-targeting, on atarget cell from (target cell 1) leads to the induction of the celldeath of the treated cell (target cell 1).

FIG. 9B: During the cell death the dying cell (target cell 1, 1)releases nuclear antigens (such as the La protein) from the nucleuswhich bind stably to the surface of the neighboring cell(s) (target cell2).

FIG. 9C: Immune effector cells (here a T cell) are recruited by abispecific antibody derivative (anti-La/anti-CD3) to the La-markedtarget cell 2. The bispecific antibody derivative is, on the one hand,directed against a peptide epitope in the La protein and binds, on theother hand, to an activating domain on the immune effector cell (hereCD3 on T cells). By binding of La protein to neighboring cells and therecruitment of effector cells to the target cells, it is advantageouslypossible to destroy tumor cells (target cell 2) which had originally nospecific target structure on the cell surface. Also, e.g. stroma cellsor endothelial cells which supply the tumor cell can be destroyed inthis manner.

FIGS. 10A-10H show the redox sensitivity of the La antigen: The antibody7B6 recognizes an oxidized variant. When human cell culture cells arefixed with methanol, the cells cannot be stained with the anti-La mAk7B6 (FIG. 10A). When the cells are washed before staining with PBScontaining H₂O₂ (FIG. 10C), then mAk 7B6 can however stain the cells.When oxidative stress is exerted on the cells before fixation (here byUV radiation), mAk 7B6 (FIG. 10E) is also able to stain the cells afterfixation. When doing so, after UV exposure, dependent on time, atranslocation into the cytoplasm (FIG. 10G) occurs. FIGS. 10B, 10D, 10F,and 10H are the corresponding phase contrast images to theepifluorescence images (FIGS. 10A, 10C, 10E, and 10G).

FIGS. 11A-11B show the stability of the rhLa protein on the cell surfaceof 3T3 cells up to 24 hrs.

The day before, mouse 3T3 cells were seeded into several 24-well plates.All cells were incubated in parallel for 1 hr. with 1.7 μM of rhLaprotein in DMEM at 37° C. and then the material that was not bound iswashed off. Untreated cells were carried along as a control.

FIG. 11A: The cells were stained either immediately (0 hrs.) on ice with5B9 or anti-His (prime. Ak) and the anti-mouse-IgG-Alexa Fluor®488-antibody, or they were transferred into medium and were cultured foranother 3 hrs or 24 hrs in an incubator before they were also stained.After staining the cells were removed with PBS-EDTA and analyzed in aflow cytometer. The cells that had been cultured longer were accordinglystained and measured later. FIG. 11B: Western blot of the total extractswhich had been produced immediately after La decoration, after 3 hrs. or24 hrs. Parallel to this, samples with cells without La loading werecarried along. In the first trace of the gel the amount of rhLa proteinwas applied which had been given also to the cells (“in”). The westernblots were developed with 5B9 and anti-His as well as anti-mouse-IgG-AP.The arrow marks the band of the rhLa protein which could be detectedwith 5B9 and anti-His. (*) indicates the endogenous mLa protein which isalso recognized by 5B9.

FIGS. 12A-12B show construction, eukaryote production as well as bindingstudies for the anti-La-scFvs.

FIG. 12A: In the anti-La-scFv molecule the variable domains of the mAks7B6 are linked with each other in the order V_(H)-V_(L) by a flexibleglycine serine linker ((G₄S)₃). The Igic signal sequence coded by thevector pSecTag2 B enables the secretion of the proteins in the medium. Ac-myc and a hexahistidine-(His₆-) tag are added C-terminally. FIG. 12B:After purification of 7B6 scFv from the cell culture supernatent,binding of the protein (10 μg) on La-decorated tumor cells was examined.The detection was carried out with anti-c-myc-FITC. Parallel to this,cells were stained with mAk 7B6 and anti-mouse-IgG-Alexa Fluor® 488.Moreover, cells were incubated with the antibodies, respectively,without prior La loading (−rhLa).

FIGS. 13A-13E show construction, purification and binding studies of thebispecific antibody CD3x7B6.

FIG. 13A: The DNA sequence of 7B6 scFv iC was cloned via BamHIrestriction sites into the vector pSecTag2 B-CD3 scFv oC in order toobtain the plasmid pSecTag2 B-CD3x7B6 scBsDb. FIG. 13B: The proteinCD3x7B6 has an N-terminal Igic signal peptide which mediates thesecretion into the cell culture medium. To the diabody are addedC-terminally a c-myc as well as hexahistidine-(His6-) tag to permit thedetection of the protein or the purification by Ni-NTA agarose. FIG.13C: Purification of the eukaryote-produced CD3x7B6 scBsDb. Fromtransduced HEK 293T CD3x7B6 scBsDb cells the supernatent (SN) wasremoved and applied to an Ni-NTA spin column. The flow (DL) wascollected. Subsequently, washing with 10 mM imidazole (W1) and 20 mMimidazole (W2) was carried out. The elution of the diabody was done with350 mM imidazole (E1-E3). A portion of the cells was taken up in 2×SDSsample buffer (pellet, P) and together with aliquots of all fractionsapplied onto a 12% polyacrylamide gel. After western blot on anitrocellulose membrane, CD3x7B6 scBsDb was detected with anti-His andanti-mouse-IgG-AP. Moreover, 10 μl of elution 1 were applied to a secondgel which was stained with Coomassie Brilliant Blue (CBB). FIG. 13D:Binding analysis on CD3⁺ T lymphocytes. As a positive control, theantibody anti-CD3-FITC (black) is shown in comparison to its isotypecontrol. Parallel to this, the PBMCs were incubated with 10 μg CD3x7B6which was detected with anti-c-myc-FITC (black). As a control,anti-c-myc-FITC was used alone. FIG. 13E: FACS analysis on HEK 293T PSCAcells which had been pretreated with 9 μM of rhLa protein (+rhLa) orwithout it (−rhLa). The detection of the La protein was done either withmAk 7B6 and anti-mouse-IgG-Alexa Fluor® 488 or with 10 μg CD3x7B6 andanti-c-myc-FITC.

FIG. 14 shows the T cell-mediated cytotoxicity in the presence ofCD3x7B6.

For the cytotoxicity test the target cells HEK 293T PSCA were loadedwith ⁵¹Cr. Then one quarter of the cells was incubated without Laprotein or with 1 μM or 30 μM of rhLa protein. Subsequently, the targetcells were co-cultured in a ratio of 1:20 with preactivated T cells.Moreover, to some of the samples 100 nM of CD3x7B6 protein was added.After 18 hrs. the chrome release was measured. The average values of atriple determination and their standard deviations are shown. Thestatistical significance was determined with the Student's t test(***p<0.001). A representative donor is shown for three examined ones.

DESCRIPTION OF PREFERRED EMBODIMENTS Example 1 Binding and Detection ofthe rhLa Protein on the Cell Surface

For the surface binding studies of the La protein different cell linesas well as PBMCs were examined. The adherent cell lines were seeded in12-well plates and on the subsequent day incubated with 2-10 μM of rhLaprotein in PBS or DMEM. The one-hour La binding took place either on iceor at 37° C. Subsequently, the cells on ice were stained with theanti-La-mAks and a suitable secondary antibody in order to detect the Laprotein on the cell surface. Then the cells were treated with PFA,Triton X-100 and DAPI solutions before immunofluorescence images weretaken. For the quantitative binding studies the stained, unfixed cellswere removed with PBS-EDTA and examined flow-cytometrically.Alternatively, the adherent cells were first removed for the flowcytometry with PBS-EDTA and then incubated in a 96-well plate with Laand the antibodies.

In the following it was checked how long after decoration the La proteincan still be detected on the cells and whether it is perhapsproteolytically cleaved and is only partially present on the cellsurface. For this purpose, mouse 3T3 cells were incubated for 1 hr. withrhLa in DMEM at 37° C. Then the medium was changed. The cells wereexamined either immediately or cultured for another 3 hrs or 24 hrs. Ateach point in time cells were stained on ice with the differentanti-La-mAks 27E, 5B9, SW5 (comparative example), 22A and 7B6 as well asanti-His and a suitable secondary antibody. Subsequently, a portion ofthe cells was removed with PBS-EDTA from the culture vessels andanalyzed in the flow cytometer (see FIG. 11A). Moreover, total extractsof the La-loaded cells and control cells without La protein wereproduced. Detection with the different antibodies was carried out bywestern blot (see FIG. 11B).

Example 2 Immunization and Preparation of Hybridomas

For the hybridoma preparation, mice (Balb/C) were immunized withrecombinant La protein (rhLa—SEQ ID No. 1 with His tag, 40 mice)produced in E. coli BL21 DE3 pLysS and purified by means ofnickel-affinity chromatography. 10 other mice were immunized withrecombinant rhLa1 192 antigen (La-peptide with amino acid residues from1 to 192 of the SEQ ID No. 1) produced also in E. coli BL21 DE3 pLysS.In the first immunization 50 μg of the respective antigen were appliedin complete Freund's adjuvant (Difco, Mich., USA). In the otherimmunizations, each carried out two weeks apart, 25 μg of antigen wereapplied in each case. The antigen was for this purpose re-suspended inincomplete adjuvant (Difco, Mich., USA). Before hybridoma fusion, theanimals had been immunized four times. After isolation of the spleencells, they were fused with myeloma cells (P3×Ag 8.653; ATCC CRL 1580)in a ratio of 1:1 to 10:1 by dropwise addition of polyethylene glycol.Subsequently, the cells were selected in HAT medium (medium containinghypoxanthine, aminopterin and thymidine). Positive hybridomas wereidentified by means of ELISA and the cells recloned several times bylimited dilution.

In case of the hybridoma fusion of hLa transgenic mice after adoptivetransfer of T cells, the protocol was modified as follows.Non-transgenic mice from the same litter were immunized as describedabove several times with rhLa. Then the spleen cells were prepared andby means of nylon wool the non-adherent cells were isolated. Thecontaminating B cells were removed by anti-B220 magnetic beads (RA3-6B2,Dynal, Oslo, Norway). The purity of the isolated cells was determined bymeans of FACS. In the embodiment, 64% of the isolated cells were CD4positive, 0.76% B220 positive. The remainder consisted of CD8 positivecells. lx 10⁷ of these cells were applied intravenously on day zero intothe tail vein of a 10 week old hLa transgenic mouse (A/J background). 21days after the adoptive transfer the spleen was removed and thehybridoma fusion was carried out as described above. By means of ELISAit had been determined before that the anti-La response is optimalbetween day 21 and day 28 after transfer. Parallel to this, T cells of anon-immunized mouse were transferred into a hLa transgenic mouse. Also,a hybridoma fusion of a control mouse, not immunized, was carried out.Anti-La hybridomas were established only from the mouse that hadreceived adoptive T cells from the La-immunized mouse.

Example 3 Analysis of Hybridoma Supernatents

Recombinant His₆ hLa protein was diluted in coating buffer (1-5 μg/ml).Of this, 100 μl were added to every well of the ELISA plate andincubated over night at 4° C. or 2 hrs at 37° C. After washing the platefive times with 200 μl of ELISA washing buffer for each well per washingstep, the remaining binding sites were saturated with 200 ₁1.1 of ELISAblocking solution, respectively, for 1 hr at 37° C. After washing again,100 μl of hybridoma supernatent was added to each well of the plate.Binding occurred for 1 hr at 37° C. Subsequently, the non-boundantibodies were removed by washing five times and 100 μl of the dilutedsecondary antibody anti-mouse-IgG-POD (1:40000 in PBS) were applied toeach well. After 1 hr at 37° C. the excess antibodies were removed againby washing. Into each well 100 μl of the substrate solution werepipetted and the plate incubated in the dark. After a distinct colordevelopment was recognizable, the reaction was terminated with 50 μl ofstop solution per well. The quantification occurred through measurementof the optical density at 450 nm (reference filter 620 nm) by means ofan ELISA plate reader.

Example 4 Investigation of the Redox-Dependent Antibody Binding

The ELISA plate was coated with 10 μg/ml rhLa over night at 4° C. Thiswas followed by oxidation (3% (v/v) H₂O₂ in PBS, 30 min, RT) orreduction (2% (v/v) β-mercapto ethanol in PBS, 30 min, RT) for some ofthe wells. After blocking the whole plate, incubation with hybridomasupernatents or patients' sera was carried out. The detection wascarried out with anti-mouse-IgG-POD or anti-human-IgG-POD.

Example 5 Production of scFv Fragments and Bispecific Antibodies

The production of scFv fragments occurred based on mAk 7B6. Based on thesequences of the V_(H) and V_(L) genes which were cloned in pGEM®-TEasy, the scFv derivative was cloned into the vector pSecTag2 B. Thesuitable recombinant protein (see schematic in FIG. 12A) contains anN-terminal Igx signal sequence which mediates the secretion of the scFvmolecules into the cell culture medium and is proteolytically cleavedthereby. The VH and VL domains adjoin it and are connected by a flexibleglycine serine linker ((G₄S)₃) with each other. To the proteins onec-myc tag as well as a hexahistidine tag are added C-terminally in orderto enable the specific detection or purification by Ni-NTA affinitychromatography. These eukaryotic expression vectors were transfectedinto HEK 293T cells. The total extracts of the cells as well as the cellculture supernatents were analyzed with respect to containedanti-La-scFv molecules by immunoblot.

The 7B6 scFv protein was purified by Ni-NTA affinity chromatography fromthe cell culture medium. The single fractions were examined by westernblot which was developed with anti-His in regard to the presence of therecombinant protein. By FACS analyses (see FIG. 12B) binding to cellsdecorated with rhLa was examined. The La protein was specifically boundby mAk 7B6 as well as by 7B6 scFv. The control cells which had not beenpretreated with rhLa protein were not stained in both cases.

Since 7B6 scFv was able to bind the rhLa protein on the cell surface, itwas used for the generation of a bispecific CD3x7B6 antibody (singlechain bispecific diabody—scBsDb). In the plasmid pSecTag2 B-CD3 scFv oCthe variable domains are organized in the sequence V_(H) CD3-VL CD3 andare connected by a flexible glycine serine linker ((G₄S)₃) with eachother. For the second antigen specificity of the diabody, the innercassette 7B6 scFv iC (see FIG. 13A, CD scFv iC) with the domain sequenceVL 7B6 (G₄S) 5-VH 7B6-G₄S was inserted into this outer cassette (seeFIG. 13A, CD scFv oC). By treatment with the restriction enzyme BamHIthe vector pSecTag2 B-CD3 scFv oCc was linearized between V_(H) CD3 andV_(L) CD3 and the DNA fragment of 7B6 scFv iC generated in parallel byBamHI was inserted by ligation (see FIG. 13A). The obtained clones werechecked by sequencing. Subsequently, the resultant plasmid pSecTag2B-CD3x7B6 scBsDb was transfected transiently in HEK 293T cells in orderto analyze the production and secretion of the CD3x7B6 diabody. As aresult of the used vector, the bispecific CD3x7B6 antibody (scBsDb)again has an N-terminal Igx signal sequence, and a c-myc tag and ahexahistidine tag adjoin the protein C-terminally (see FIG. 13B). Inorder to have at disposal sufficient protein amounts for otherexperiments, a stable cell line was established by transduction thatcontinuously secrets CD3x7B6 protein. An amplification of the CD3x7B6DNA sequence by PCR was necessary for this purpose in order to addN-terminally an EcoRI restriction site and C-terminally a Kpn2Irestriction site. Finally, by these two restriction enzymes cloningoccurred into the retroviral expression vector pczCFG5.1. For thetransduction HEK 293T cells were also used as target cells.

The transiently transfected as well as stably transduced HEK 293T cellswere capable of producing the CD3x7B6 scBsDb protein and to release itinto the cell culture medium. From the latter, it could be obtained byNi-NTA affinity chromatography (see FIG. 13C). Binding of the protein toCD3+ T lymphocytes was examined on human PBMCs (see FIG. 13D). Theprotein as well as the antibody anti-CD3-FITC used as a positive controlwas detected on 74% of the lymphocytes. This proved the functionality ofthe CD3 arm of the diabody.

Moreover, binding of the CD3x7B6 molecule on rhLa on the cell surfacewas analyzed. The 7B6 side of the diabody was able to recognize 98% ofthe cells. This corresponded nearly to 100% of the mAks 7B6 which causedby its bivalent binding, as expected, a stronger shift of the whole cellpopulation in the green fluorescence channel (see FIG. 13E).

Since the CD3x7B6 protein could be detected on La-decorated tumor cellsas well as on CD3+ T cells, it can cause a cross-linking between tumorcells and T-effector cells in co-culturing of both cell populations. Thecytotoxic T cells are thereby activated and, as a result, the targetcells are lysed by them (see schematic in FIG. 9). In order to clarifythese effector mechanisms, a chrome release test was carried out. Thetumor cells after chrome loading were incubated with different amountsof rhLa protein. Subsequently, they were co-cultured for 18 hrs withpreactivated T cells. These T cells are predominantly CD8+ cytotoxic Tcells which were obtained by incubation of PBMCs with IL-2 and anti-CD3(OKT3). This protocol was developed at the Institut für Immunologie andthe obtained T cells have been characterized in detail. The results ofthe chrome release test are shown in FIG. 14.

For the cytotoxicity test the target cells HEK 293T PSCA were loadedwith ⁵¹Cr. Then one quarter of the cells was incubated without Laprotein or with 1 μM, 10 μM or 30 μM of rhLa protein, respectively.Subsequently, the target cells were co-cultured in a ratio 1:20 withpreactivated T cells. Moreover, to a part of the batches 100 nM ofCD3x7B6 protein was added. After 18 hrs the chrome release was measured.The average values of a triple determination and their standarddeviations are shown. The statistical significance was determined byStudent's t test (*** p<0.001). A representative donor is shown forthree examined ones.

What is claimed is:
 1. Recombinant antibody, comprising (a) a bindingunit of a first antibody that specifically binds to an epitope of ahuman nuclear antigen, wherein one such human nuclear antigen cancomprise human La, characterized in that the first antibody is anantibody which binds specifically an epitope of the human La protein(anti-La antibody) containing regions determining complementarity(complementarity determining regions, CDRs), the CDRs of the variableregion of the light chain (V L) and the variable region of the heavychain (V H) comprising the following sequences: SEQ SEQ ID ID V_(L) No.V_(H) No. CDR1 RSSQSLLDSRTRKNYLA 9 DFWMN 10 CDR2 WASTRES 11QIRNKPNNYETYYSDSLKG 12 CDR3 KQSYNLPT 13 LGNSWFAY 14

and (b) a binding unit comprising: a second antibody which bindsspecifically to an effector cell or a ligand which binds specifically toan effector cell.
 2. The recombinant antibody according to claim 1,characterized in that the second antibody or the ligand which bindsspecifically to an effector cell is selected from antibodies and ligandswhich bind specifically surface structures on T lymphocytes, NK cellsand/or monocytes.
 3. The recombinant antibody according to claim 1containing the following, optionally humanized, structure: a variableregion of the light chain having a sequence of SEQ ID No. 53 and avariable region of the heavy chain having a sequence of SEQ ID No. 54.4. The recombinant antibody according to claim 1, in the form of a scFvfragment, a F(ab′)₂ fragment, diabodies, triabodies or tetrabodies. 5.Antibodies which bind specifically an epitope of the human La protein(anti-La antibody) containing regions determining complementarity(complementarity determining regions, CDRs), characterized in that theCDRs of the variable region of the light chain (V_(L)) and the variableregion of the heavy chain (V_(H)) comprise the following sequences: SEQSEQ ID ID V_(L) No. V_(H) No. CDR1 RSSQSLLDSRTRKNYLA 9 DFWMN 10 CDR2WASTRES 11 QIRNKPNNYETYYSDSLKG 12 CDR3 KQSYNLPT 13 LGNSWFAY 14


6. The antibodies according to claim 5, containing the following,optionally humanized, structure: a variable region of the light chainhaving a sequence of SEQ ID No. 53 and a variable region of the heavychain having a sequence of SEQ ID No.
 54. 7. The antibodies according toclaim 5 in the form of a scFv fragment, a F(ab′)₂ fragment, diabodies,triabodies or tetrabodies.